Design, synthesis, and biological evaluation of 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides as a potential EGR-1 inhibitor for targeted therapy of atopic dermatitis.

Atopic dermatitis is a chronic inflammatory skin disease characterized by intense itching and frequent skin barrier dysfunctions. EGR-1 is a transcription factor that aggravates the pathogenesis of atopic dermatitis by promoting the production of various inflammatory cytokines. Three 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamides (IT21, IT23, and IT25) were identified as novel inhibitors of EGR-1 DNA-binding activity. In silico docking experiments were performed to elucidate the binding conditions of the EGR-1 zinc-finger (ZnF) DNA-binding domain. Electrophoretic mobility shift assays confirmed the targeted binding effect on the EGR-1 ZnF DNA-binding domain, leading to dose-dependent dissociation of the EGR-1-DNA complex. At the functional cellular level, IT21, IT23, and IT25 effectively reduced mRNA expression of TNFα-induced EGR-1-regulated inflammatory genes, particularly in HaCaT keratinocytes inflamed by TNFα. In the in vivo efficacy study, IT21, IT23, and IT25 demonstrated the potential to alleviate atopic dermatitis-like skin lesions in the ear skin of BALB/c mice. These findings suggest that targeting the EGR-1 ZnF DNA-binding domain with 2-(2-oxoindolin-3-ylidene)hydrazinecarbothioamide derivatives (IT21, IT23, and IT25) could serve as lead compounds for the development of potential therapeutic agents against inflammatory skin disorders, including atopic dermatitis.

Lipid Emulsions Inhibit Labetalol-Induced Vasodilation in the Isolated Rat Aorta.

Lipid emulsions are used as adjuvant drugs to alleviate intractable cardiovascular collapse induced by drug toxicity. We aimed to examine the effect of lipid emulsions on labetalol-induced vasodilation and the underlying mechanism in the isolated rat aorta. We studied the effects of endothelial denudation, NW-nitro-l-arginine methyl ester (l-NAME), calmidazolium, methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ), and lipid emulsions on labetalol-induced vasodilation. We also evaluated the effects of lipid emulsions on cyclic guanosine monophosphate (cGMP) formation, endothelial nitric oxide synthase (eNOS) phosphorylation, and endothelial calcium levels induced by labetalol. Labetalol-induced vasodilation was higher in endothelium-intact aortas than that in endothelium-denuded aortas. l-NAME, calmidazolium, methylene blue, and ODQ inhibited labetalol-induced vasodilation in endothelium-intact aortas. Lipid emulsions inhibited labetalol-induced vasodilation in endothelium-intact and endothelium-denuded aortas. l-NAME, ODQ, and lipid emulsions inhibited labetalol-induced cGMP formation in endothelium-intact aortas. Lipid emulsions reversed the stimulatory and inhibitory eNOS (Ser1177 and Thr495) phosphorylation induced by labetalol in human umbilical vein endothelial cells and inhibited the labetalol-induced endothelial calcium increase. Moreover, it decreased labetalol concentration. These results suggest that lipid emulsions inhibit vasodilation induced by toxic doses of labetalol, which is mediated by the inhibition of endothelial nitric oxide release and reduction of labetalol concentration.

Hypothermia Inhibits Dexmedetomidine-Induced Contractions in Isolated Rat Aortae.

Dexmedetomidine is widely used to induce sedation in the perioperative period. This study examined the effect of hypothermia (33 and 25 °C) on dexmedetomidine-induced contraction in an endothelium-intact aorta with or without the nitric oxide synthase inhibitor NW-nitro-L-arginine methyl ester (L-NAME). In addition, the effect of hypothermia on the contraction induced by dexmedetomidine in an endothelium-denuded aorta with or without a calcium-free Krebs solution was examined. The effects of hypothermia on the protein kinase C (PKC), myosin light chain (MLC20) phosphorylation, and Rho-kinase membrane translocation induced by dexmedetomidine were examined. Hypothermia inhibited dexmedetomidine-induced contraction in the endothelium-intact aorta with L-NAME or endothelium-denuded aorta. Hypothermia had almost no effect on the dexmedetomidine-induced contraction in the endothelium-denuded aorta with the calcium-free Krebs solution; however, the subsequent contraction induced by the addition of calcium was inhibited by hypothermia. Conversely, the transition from profound hypothermia back to normothermia reversed the hypothermia-induced inhibition of subsequent calcium-induced contractions. Hypothermia inhibited any contraction induced by KCl, PDBu, and NaF, as well as PKC and MLC20 phosphorylation and Rho-kinase membrane translocation induced by dexmedetomidine. These results suggest that hypothermia inhibits dexmedetomidine-induced contraction, which is mediated mainly by the impediment of calcium influx and partially by the attenuation of pathways involving PKC and Rho-kinase activation.

Design, synthesis, and biological activities of 3-((4,6-diphenylpyrimidin-2-ylamino)methylene)-2,3-dihydrochromen-4-ones.

Novel (Z)-3-((4,6-diphenylpyrimidin-2-ylamino)methylene)-2,3-dihydrochromen-4-one derivatives were designed and synthesized to find chemotherapeutic agents. Derivative 9 was selected based on its clonogenicity against cancer cells and synthetic yield for further biological experiments. It showed decreases in aurora kinase A, B, and C phosphorylation from western blot analysis. Derivative 9 upregulated the expression of G1 cell cycle inhibitory proteins including p21 and p27, and G1 progressive cyclin D1, and downregulated G1-to-S progressive cyclins, resulting in cell cycle arrest at the G1/S boundary. It stimulated the cleavage of caspase-9, -3, -7, and poly (ADP-ribose) polymerase, resulting in triggering apoptosis through a caspase-dependent pathway. In addition, derivative 9 inhibited in vivo tumor growth in a syngeneic tumor implantation mouse model. The findings of this study suggest that derivative 9 can be considered as a lead compound for chemotherapeutic agents.

A Novel Synthetic Compound (E)-5-((4-oxo-4H-chromen-3-yl)methyleneamino)-1-phenyl-1H-pyrazole-4-carbonitrile Inhibits TNFα-Induced MMP9 Expression via EGR-1 Downregulation in MDA-MB-231 Human Breast Cancer Cells.

Breast cancer is a common malignancy among women worldwide. Gelatinases such as matrix metallopeptidase 2 (MMP2) and MMP9 play crucial roles in cancer cell migration, invasion, and metastasis. To develop a novel platform compound, we synthesized a flavonoid derivative, (E)-5-((4-oxo-4H-chromen-3-yl)methyleneamino)-1-phenyl-1H-pyrazole-4-carbonitrile (named DK4023) and characterized its inhibitory effects on the motility and MMP2 and MMP9 expression of highly metastatic MDA-MB-231 breast cancer cells. We found that DK4023 inhibited tumor necrosis factor alpha (TNFα)-induced motility and F-actin formation of MDA-MB-231 cells. DK4023 also suppressed the TNFα-induced mRNA expression of MMP9 through the downregulation of the TNFα-extracellular signal-regulated kinase (ERK)/early growth response 1 (EGR-1) signaling axis. These results suggest that DK4023 could serve as a potential platform compound for the development of novel chemopreventive/chemotherapeutic agents against invasive breast cancer.

Inclusion complexes of cysteinyl ß-cyclodextrin with baicalein restore collagen synthesis in fibroblast cells following ultraviolet exposure.

Baicalein, a bioactive flavonoid, has poor water solubility, thereby limiting its use in a wide range of biological applications. In the present study, we used inclusion complexes of cysteinyl ß-cyclodextrin (ß-CD) with baicalein to enhance the stability and solubility of baicalein in aqueous solution. We examined the effects of inclusion complexes of cysteinyl ß-CD on collagen synthesis following ultraviolet (UV) irradiation, as well as the mechanisms underlying its effects. Our findings demonstrated that baicalein significantly restored collagen synthesis in the UV-exposed human fibroblast Hs68 cells. In addition, synthetic cysteine functionalized ß-CDs were found to promote baicalein-induced collagen synthesis. Inclusion complexes of cysteinyl ß-CDs with baicalein significantly upregulated the protein expression of type I collagen and activated the transcription of type I, II, and III collagen. Inclusion complexes of cysteinyl ß-CDs with baicalein also downregulated matrix metalloproteinase -1 and -3, and α-smooth muscle actin expression. In addition, inclusion complexes of cysteinyl ß-CDs with baicalein attenuated the expression of caveolin-1, but this treatment enhanced the UV-induced phosphorylation of Smad in the transforming growth factor-ß pathway. These results suggested that the newly synthesized derivative of CD can be used as a complexing agent to enhance the bioavailability of flavonoids such as baicalein, especially in restoring collagen synthesis.

Design, synthesis, and biological activities of 1-aryl-(3-(2-styryl)phenyl)prop-2-en-1-ones.

A moderate elevation in reactive oxygen species (ROS) levels can generally be controlled in normal cells, but may lead to death of cancer cells as the ROS level in cancer cells is already elevated. Therefore, a ROS-generating compound can act as a selective chemotherapeutic agent for cancer cells that does not affect normal cells. In our previous study, a compound containing a Michael acceptor was selectively cytotoxic to cancer cells without affecting normal cells; therefore, we designed and synthesized 26 compounds containing a Michael acceptor. Their cytotoxicities against HCT116 human colon cancer cell lines were measured by using a clonogenic long-term survival assay. To derive the structural conditions required to obtain stronger cytotoxicity against cancer cells, the relationships between the half-maximal cell growth inhibitory concentration values of the synthesized compounds and their physicochemical properties were evaluated by Comparative Molecular Field Analysis and Comparative Molecular Similarity Indices Analysis. It was confirmed that the compound with the best half-maximal cell growth inhibitory concentration triggered apoptosis through ROS generation, which then led to stimulation of the caspase pathway.

Inhibitory Effect of Synthetic Flavone Derivatives on Pan-Aurora Kinases: Induction of G2/M Cell-Cycle Arrest and Apoptosis in HCT116 Human Colon Cancer Cells.

Members of the aurora kinase family are Ser/Thr kinases involved in regulating mitosis. Multiple promising clinical trials to target aurora kinases are in development. To discover flavones showing growth inhibitory effects on cancer cells, 36 flavone derivatives were prepared, and their cytotoxicity was measured using a long-term clonogenic survival assay. Their half-maximal growth inhibitory effects against HCT116 human colon cancer cells were observed at the sub-micromolar level. Pharmacophores were derived based on three-dimensional quantitative structure⁻activity calculations. Because plant-derived flavones inhibit aurora kinase B, we selected 5-methoxy-2-(2-methoxynaphthalen-1-yl)-4H-chromen-4-one (derivative 31), which showed the best half-maximal cell growth inhibitory effect, and tested whether it can inhibit aurora kinases in HCT116 colon cancer cells. We found that derivative 31 inhibited the phosphorylation of aurora kinases A, aurora kinases B and aurora kinases C, suggesting that derivative 31 is a potential pan-aurora kinase inhibitor. The results of our analysis of the binding modes between derivative 31 and aurora A and aurora B kinases using in-silico docking were consistent with the pharmacophores proposed in this study.

Biological evaluation of 2-pyrazolinyl-1-carbothioamide derivatives against HCT116 human colorectal cancer cell lines and elucidation on QSAR and molecular binding modes.

In the search of compounds exhibiting anticancer activity, 37 derivatives of 2-pyrazolinyl-1-carbothioamide were designed and synthesized. Clonogenic cell survival assays were adapted to measure the cytotoxicities of the synthetic derivatives against HCT116 human colon cancer cell lines. Half-maximal cell growth inhibitory concentrations (GI50) ranged from 0.49 to 41.22µM. The compound with the lowest GI50 value, 3-(2-hydroxy-4,5-dimethoxyphenyl)-5-(naphthalen-1-yl)-N-(3,4,5-trimethoxyphenyl)-pyrazolinyl-1-carbothioamide, was subjected to further biological studies, including cell viability and apoptosis assays to examine levels of annexin-V in the outer plasma membrane layer and poly ADP-ribose polymerase cleavage. Additionally, in vitro kinase assays were performed, and Abelson murine leukemia viral oncogene homolog 1 (Abl 1) tyrosine kinase demonstrated good inhibitory activity. The binding mode between the compound of interest and Abl 1 was elucidated using in silico docking. The pharmacophores derived for 2-pyrazolinyl-1-carbothioamides based on their quantitative structure-activity relationships will help us design novel chemotherapeutic agents.

p53-dependent and -independent mechanisms are involved in (E)-1-(2-hydroxyphenyl)-3-(2-methoxynaphthalen-1-yl)prop-2-en-1-one (HMP)-induced apoptosis in HCT116 colon cancer cells.

(E)-1-(2-hydroxyphenyl)-3-(2-methoxynaphthalen-1-yl)prop-2-en-1-one (HMP) is a novel synthetic naphthal chalcone derivative. The aim of this study was to investigate the mode of action underlying the antitumor activity of HMP. We found that treatment with HMP potently inhibited the clonogenicity and triggered cell death in HCT116 colon cancer cells. Flow cytometry showed that HMP induced an increase in the population of sub-G0/G1-phase cells. Annexin V binding assay revealed that HMP triggered apoptotic cell death. Furthermore, HMP stimulated the cleavages of caspase-7 and its substrate poly (ADP-ribose) polymerase (PARP). HMP promoted γ-H2AX formation and the production of reactive oxygen species (ROS), and up-regulated expression of the tumor suppressor p53. Interestingly, HMP-induced caspase-7 processing was not completely abrogated in p53-null (p53-/-) HCT116 cells, suggesting that p53-dependent and -independent mechanisms are involved in HMP-induced apoptosis. Egr-1, a zinc finger transcription factor, was upregulated by HMP. Silencing of Egr-1 by shRNA significantly reduced HMP-induced caspase-7 and PARP cleavages, regardless of p53 status. These results suggest that HMP triggers caspase-mediated apoptosis through two distinct mechanisms involving p53-dependent and p53-independent, Egr-1-dependent pathways.

Colorectal anticancer activities of polymethoxylated 3-naphthyl-5-phenylpyrazoline-carbothioamides.

To develop potent chemotherapeutic agents for treating colorectal cancers, polymethoxylated 3-naphthyl-5-phenylpyrazoline-carbothioamide derivatives were designed. Twenty-two novel derivatives were synthesized and their cytotoxicities were measured using a clonogenic long-term survival assay. Of these derivatives, 3-(1-hydroxynaphthalen-2-yl)-N-(3-methoxyphenyl)-5-(4-methoxyphenyl)-pyrazoline-1-carbothioamide (NPC 15) exhibited the best half-maximal cell growth inhibitory concentrations (196.35nM). To explain its cytotoxicity, further biological experiments were performed. Treatment with NPC 15 inhibited cell cycle progression and triggered apoptosis through the caspase-mediated pathway. Its inhibitory effects on several kinases participating in the cell cycle were investigated using an in vitro kinase assay. Its half-maximal inhibitory concentrations for aurora kinases A and B were 105.03µM and 8.53µM, respectively. Further analysis showed that NPC 15 decreased phosphorylation of aurora kinases A, B, and C and phosphorylation of histone H3, a substrate of aurora kinases A and B. Its molecular binding mode for aurora kinase B was elucidated using in silico docking. In summary, polymethoxylated 3-naphthyl-5-phenylpyrazoline-carbothioamides could be potent chemotherapeutic agents.

Anticancer and structure-activity relationship evaluation of 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide analogs of chalcone.

To identify new potent chemotherapeutic agents, we synthesized compounds with 3-(naphthalen-2-yl)-N,5-diphenyl-pyrazoline-1-carbothioamide (NDPC) skeletons and evaluated their cytotoxicities using a clonogenic long-term survival assay. Their half-maximal cell growth inhibitory concentrations ranged from a few hundred nanomolars to a few micromolars. Further biological experiments including flow cytometry and western blotting analysis were performed with the derivative showing the best cytotoxicity. To identify a target protein of the selected compound, an in vitro kinase assay was carried out, which revealed that aurora kinases A and B were inhibited by the test compound, and this was confirmed using western blot analysis. The molecular binding mode between the selected compound and the kinases was elucidated using in silico docking. The structural conditions required for good cytotoxicity were identified based on the quantitative relationships between the physicochemical properties of the derivatives and their cytotoxicities.

Polyphenols bearing cinnamaldehyde scaffold showing cell growth inhibitory effects on the cisplatin-resistant A2780/Cis ovarian cancer cells.

Ovarian carcinoma remains the most lethal among gynecological cancers. Chemoresistance is a clinical problem that severely limits treatment success. To identify potent anticancer agents against the cisplatin-resistant human ovarian cancer cell line A2780/Cis, 26 polyphenols bearing a cinnamaldehyde scaffold were synthesized. Structural differences in their inhibitory effect on clonogenicity of A2780/Cis cells were elucidated using comparative molecular field analysis and comparative molecular similarity indices analysis. Structural conditions required for increased inhibitory activity can be derived based on the analysis of their contour maps. The two most active compounds (16 and 19) were selected and further characterized their biological activities. We found that compounds 16 and 19 trigger cell cycle arrest at the G2/M phase and apoptotic cell death in cisplatin-resistant A2780/Cis human ovarian cancer cells. The molecular mechanism of compound 16 was elucidated using in vitro aurora A kinase assay, and the binding mode between the compound 16 and aurora A kinase was interpreted using in silico docking experiments. The findings obtained here may help us develop novel plant-derived polyphenols used for potent chemotherapeutic agents. In conclusion, compounds 16 and 19 could be used as promising lead compounds for the development of novel anticancer therapies in the treatment of cisplatin-resistant ovarian cancers.

Butein derivatives prevent obesity and improve insulin resistance through the induction of energy expenditure in high-fat diet-fed obese mice.

Obesity is a global public health problem and is related with fatal diseases such as cancer and cardiovascular and metabolic diseases. Medical and lifestyle-related strategies to combat obesity have their limitations. White adipose tissue (WAT) browning is a promising strategy for increasing energy expenditure in individuals with obesity. Uncoupling protein 1 (UCP1) drives WAT browning. We previously screened natural products that enable induction of Ucp1 and demonstrated that these natural products induced WAT browning and increased energy expenditure in mice with diet-induced obesity. In this study, we aimed to extensively optimise the structure of compound 1, previously shown to promote WAT browning. Compound 3 s exhibited a significantly higher ability to induce Ucp1 in white and brown adipocytes than did compound 1. A daily injection of compound 3 s at 5 mg/kg prevented weight gain by 13.6 % in high-fat diet-fed mice without any toxicological observation. In addition, compound 3 s significantly improved glucose homeostasis, decreased serum triacylglycerol levels, and reduced total cholesterol and LDL cholesterol levels, without altering dietary intake or physical activity. Pharmaceutical properties such as solubility, lipophilicity, and membrane permeability as well as metabolic stability, half-life (T1/2), and blood exposure ratio of i.p to i.v were significantly improved in compound 3 s when compared with those in compound 1. Regarding the mode of action of WAT browning, the induction of Ucp1 and Prdm4 by compounds 1 and 3 s was dependent on Akt1 in mouse embryonic fibroblasts. Therefore, this study suggests the potential of compound 3 s as a therapeutic agent for individuals with obesity and related metabolic diseases, which acts through the induction of WAT browning as well as brown adipose tissue activation.

Benzochalcones bearing pyrazoline moieties show anti-colorectal cancer activities and selective inhibitory effects on aurora kinases.

Colorectal cancer is the third and fourth leading cause of cancer in males and females, respectively. Flavonoids, including chalcones, are secondary metabolites in plants that exhibit diverse biological activities, including antibacterial, antimalarial, and antitumor activities. In order to find potent and novel chemotherapy drugs for colorectal cancer, a series of benzochalcone derivatives, in which an α,ß-unsaturated carbonyl group was replaced with a pyrazoline, was designed and synthesized. A clonogenic survival assay was performed with each derivative to evaluate antitumor activity. 1-(5-(2,4-Dimethoxyphenyl)-4,5-dihydro-1H-pyrazol-3-yl)naphthalen-2-ol (derivative 7) had the most potent inhibitory effect on the long-term clonogenicity of HCT116 human colorectal cancer cells (IC50=2.4 µM). The results of Western blot and flow cytometric analyses suggested that derivative 7 could inhibit the proliferation of colorectal cancer cells through inhibition of cell cycle progression and induction of apoptosis. To elucidate its molecular mechanism, in vitro kinase binding assays were carried out, which demonstrated that derivative 7 inhibited aurora kinases A and B selectively. The binding modes between the compound and aurora kinases were interpreted using in silico docking experiments to explain the selective inhibitory effects on aurora kinases A and B. These findings will facilitate the design of potent novel benzochalcones as anticancer agents.

Chromenylchalcones showing cytotoxicity on human colon cancer cell lines and in silico docking with aurora kinases.

Due to toxicity problems, various plant-derived compounds have been screened to find the chemotherapeutic agents. As anticancer therapeutic agents, chalcones have advantages such as poor interaction with DNA and low risk of mutagenesity. Chromenones show anticancer activities too. Therefore, hybrids of chalcone and chromenone may be potent chemotherapeutic agents. We prepared 16 synthetic chromenylchalcones and applied a clonogenic long-term survival assay method for them on HCT116 human colorectal cancer cell lines. One of chromenylchalcones tested here, chromenylchalcone 11, showed IC50 of 93.1nM which can be competed with the IC50 values of well-known flavonoids such as catechin gallate and epicatechin gallate. Further biological experiments including cell cycle analysis, apoptosis assay, Western blot analysis, and immunofluorescent microscopy were carried out for this compound. In addition, in vitro kinases binding assay performed to explain its molecular mechanism demonstrated the compound inhibited aurora kinases. The binding modes between chromenylchalcone 11 and aurora kinases were elucidated using in silico docking experiments. These findings could be used for designing cancer therapeutic or preventive plant-derived chromenylchalcone agents.

Chromenylchalcones with inhibitory effects on monoamine oxidase B.

Structure-activity relationship (SAR) calculations were used to find monoamine oxidase-B (MAO-B) inhibitors by identifying pharmacophores exhibiting high inhibitory activities. Several such chromenylchalcones were designed and synthesized accordingly. Their inhibitory effects on MAO-B were determined using an HPLC-based method and an MAO-B enzyme assay kit. (E)-3-(6-Methoxy-2H-chromen-3-yl)-1-(2-methoxyphenyl)prop-2-en-1-one exhibited a half-maximal inhibitory concentration of 320 nM. Its molecular-level binding mode with the three-dimensional structure of MAO-B was elucidated using an in silico docking study. The chromenylchalcone scaffold, which is derived from natural products including isoflavonoids and chalcones, had not been previously reported as an MAO-B inhibitor.